The pro-inflammatory signature of lipopolysaccharide in spontaneous contracting embryoid bodies differentiated from mouse embryonic stem cells.

Embryonic stem (ES) cells differentiate towards all three germ layers, including cardiac cells and leukocytes, and may be therefore suitable to model inflammatory reactions in vitro. In the present study, embryoid bodies differentiated from mouse ES cells were treated with increasing doses of lipopolysaccharide (LPS) to mimic infection with gram-negative bacteria. LPS treatment dose-dependent increased contraction frequency of cardiac cell areas and calcium spikes and increased protein expression of α-actinin. LPS treatment increased the expression of the macrophage marker CD68 and CD69, which is upregulated after activation on T cells, B cells and NK cells. LPS dose-dependent increased protein expression of toll-like receptor 4 (TLR4). Moreover, upregulation of NLR family pyrin domain containing 3 (NLRP3), IL-1ß and cleaved caspase 1 was observed, indicating activation of inflammasome. In parallel, generation of reactive oxygen species (ROS), nitric oxide (NO), and expression of NOX1, NOX2, NOX4 and eNOS occurred. ROS generation, NOX2 expression and NO generation were downregulated by the TLR4 receptor antagonist TAK-242 which abolished the LPS-induced positive chronotropic effect of LPS. In conclusion, our data demonstrate that LPS induced a pro-inflammatory cellular immune response in tissues derived from ES cells, recommending the in vitro model of embryoid bodies for inflammation research.

Generation of a Syngeneic Heterozygous ACVRL1(wt/mut) Knockout iPS Cell Line for the In Vitro Study of HHT2-Associated Angiogenesis.

Hereditary hemorrhagic telangiectasia (HHT) type 2 is an autosomal dominant disease in which one allele of the ACVRL1 gene is mutated. Patients exhibit disturbances in TGF-beta/BMP-dependent angiogenesis and, clinically, often present with severe nosebleeds as well as a reduced quality of life. The aim of our study was to use CRISPR/Cas9 to knockout ACVRL1 in normal induced pluripotent stem cells (iPSCs) and evaluate the effects on TGF-beta- and BMP-related gene expression as well as angiogenesis. The CRISPR/Cas9 knockout of the ACVRL1 gene was carried out in previously characterized wild-type (ACVRL1wt/wt) iPSCs. An HHT type 2 iPS cell line was generated via a single-allele knockout (ACVRL1wt/mut) in wild-type (ACVRL1wt/wt) iPSCs, resulting in a heterozygous 17 bp frameshift deletion in the ACVRL1 gene [NG_009549.1:g.13707_13723del; NM_000020.3:c.1137_1153del]. After the generation of embryoid bodies (EBs), endothelial differentiation was induced via adding 4 ng/mL BMP4, 2% B27, and 10 ng/mL VEGF. Endothelial differentiation was monitored via immunocytochemistry. An analysis of 151 TGF-beta/BMP-related genes was performed via RT-qPCR through the use of mRNA derived from single iPS cell cultures as well as endothelial cells derived from EBs after endothelial differentiation. Differential TGF-beta/BMP gene expression was observed between ACVRL1wt/wt and ACVRL1wt/mut iPSCs as well as endothelial cells. EBs derived from CRISPR/Cas9-designed ACVRL1 mutant HHT type 2 iPSCs, together with their isogenic wild-type iPSC counterparts, can serve as valuable resources for HHT type 2 in vitro studies.

Loss and Recovery of Glutaredoxin 5 Is Inducible by Diet in a Murine Model of Diabesity and Mediated by Free Fatty Acids In Vitro.

Free fatty acids (FFA), hyperglycemia, and inflammatory cytokines are major mediators of ß-cell toxicity in type 2 diabetes mellitus, impairing mitochondrial metabolism. Glutaredoxin 5 (Glrx5) is a mitochondrial protein involved in the assembly of iron-sulfur clusters required for complexes of the respiratory chain. We have provided evidence that islet cells are deprived of Glrx5, correlating with impaired insulin secretion during diabetes in genetically obese mice. In this study, we induced diabesity in C57BL/6J mice in vivo by feeding the mice a high-fat diet (HFD) and modelled the diabetic metabolism in MIN6 cells through exposure to FFA, glucose, or inflammatory cytokines in vitro. qRT-PCR, ELISA, immunohisto-/cytochemistry, bioluminescence, and respirometry were employed to study Glrx5, insulin secretion, and mitochondrial biomarkers. The HFD induced a depletion of islet Glrx5 concomitant with an obese phenotype, elevated FFA in serum and reactive oxygen species in islets, and impaired glucose tolerance. Exposure of MIN6 cells to FFA led to a loss of Glrx5 in vitro. The FFA-induced depletion of Glrx5 coincided with significantly altered mitochondrial biomarkers. In summary, we provide evidence that Glrx5 is regulated by FFA in type 2 diabetes mellitus and is linked to mitochondrial dysfunction and blunted insulin secretion.

Induction of Stem-Cell-Derived Cardiomyogenesis by Fibroblast Growth Factor 10 (FGF10) and Its Interplay with Cardiotrophin-1 (CT-1).

For heart regeneration purposes, embryonic stem cell (ES)-based strategies have been developed to induce the proliferation of cardiac progenitor cells towards cardiomyocytes. Fibroblast growth factor 10 (FGF10) contributes to cardiac development and induces cardiomyocyte differentiation in vitro. Yet, among pro-cardiogenic factors, including cardiotrophin-1 (CT-1), the hyperplastic function of FGF10 in cardiomyocyte turnover remains to be further characterized. We investigated the proliferative effects of FGF10 on ES-derived cardiac progenitor cells in the intermediate developmental stage and examined the putative interplay between FGF10 and CT-1 in cardiomyocyte proliferation. Mouse ES cells were treated with FGF10 and/or CT-1. Differential expression of cardiomyocyte-specific gene markers was analyzed at transcript and protein levels. Substantial upregulation of sarcomeric α-actinin was detected by qPCR, flow cytometry, Western blot and immunocytochemistry. FGF10 enhanced the expression of other structural proteins (MLC-2a, MLC-2v and TNNT2), transcriptional factors (NKX2-5 and GATA4), and proliferation markers (Aurora B and YAP-1). FGF10/CT-1 co-administration led to an upregulation of proliferation markers, suggesting the synergistic potential of FGF10 + CT-1 on cardiomyogenesis. In summary, we provided evidence that FGF10 and CT-1 induce cardiomyocyte structural proteins, associated transcription factors, and cardiac cell proliferation, which could be applicable in therapies to replenish damaged cardiomyocytes.

Extracellular and Intracellular Angiotensin II Regulate the Automaticity of Developing Cardiomyocytes via Different Signaling Pathways.

Angiotensin II (Ang II) plays an important role in regulating various physiological processes. However, little is known about the existence of intracellular Ang II (iAng II), whether iAng II would regulate the automaticity of early differentiating cardiomyocytes, and the underlying mechanism involved. Here, iAng II was detected by immunocytochemistry and ultra-high performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry in mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) and neonatal rat ventricular myocytes. Expression of AT1R-YFP in mESC-CMs revealed that Ang II type 1 receptors were located on the surface membrane, while immunostaining of Ang II type 2 receptors (AT2R) revealed that AT2R were predominately located on the nucleus and the sarcoplasmic reticulum. While extracellular Ang II increased spontaneous action potentials (APs), dual patch clamping revealed that intracellular delivery of Ang II or AT2R activator C21 decreased spontaneous APs. Interestingly, iAng II was found to decrease the caffeine-induced increase in spontaneous APs and caffeine-induced calcium release, suggesting that iAng II decreased spontaneous APs via the AT2R- and ryanodine receptor-mediated pathways. This is the first study that provides evidence of the presence and function of iAng II in regulating the automaticity behavior of ESC-CMs and may therefore shed light on the role of iAng II in fate determination.

The Multifunctional Contribution of FGF Signaling to Cardiac Development, Homeostasis, Disease and Repair.

The current focus on cardiovascular research reflects society's concerns regarding the alarming incidence of cardiac-related diseases and mortality in the industrialized world and, notably, an urgent need to combat them by more efficient therapies. To pursue these therapeutic approaches, a comprehensive understanding of the mechanism of action for multifunctional fibroblast growth factor (FGF) signaling in the biology of the heart is a matter of high importance. The roles of FGFs in heart development range from outflow tract formation to the proliferation of cardiomyocytes and the formation of heart chambers. In the context of cardiac regeneration, FGFs 1, 2, 9, 16, 19, and 21 mediate adaptive responses including restoration of cardiac contracting rate after myocardial infarction and reduction of myocardial infarct size. However, cardiac complications in human diseases are correlated with pathogenic effects of FGF ligands and/or FGF signaling impairment. FGFs 2 and 23 are involved in maladaptive responses such as cardiac hypertrophic, fibrotic responses and heart failure. Among FGFs with known causative (FGFs 2, 21, and 23) or protective (FGFs 2, 15/19, 16, and 21) roles in cardiac diseases, FGFs 15/19, 21, and 23 display diagnostic potential. The effective role of FGFs on the induction of progenitor stem cells to cardiac cells during development has been employed to boost the limited capacity of postnatal cardiac repair. To renew or replenish damaged cardiomyocytes, FGFs 1, 2, 10, and 16 were tested in (induced-) pluripotent stem cell-based approaches and for stimulation of cell cycle re-entry in adult cardiomyocytes. This review will shed light on the wide range of beneficiary and detrimental actions mediated by FGF ligands and their receptors in the heart, which may open new therapeutic avenues for ameliorating cardiac complications.

Sexual Regional Dimorphism of Post-Adolescent and Middle Age Brain Maturation. A Multi-center 3T MRI Study.

Sex-related differences are tied into neurodevelopmental and lifespan processes, beginning early in the perinatal and developmental phases and continue into adulthood. The present study was designed to investigate sexual dimorphism of changes in gray matter (GM) volume in post-adolescence, with a focus on early and middle-adulthood using a structural magnetic resonance imaging (MRI) dataset of healthy controls from the European Network on Psychosis, Affective disorders and Cognitive Trajectory (ENPACT). Three hundred and seventy three subjects underwent a 3.0 T MRI session across four European Centers. Age by sex effects on GM volumes were investigated using voxel-based morphometry (VBM) and the Automated Anatomical Labeling atlas regions (ROI). Females and males showed overlapping and non-overlapping patterns of GM volume changes during aging. Overlapping age-related changes emerged in bilateral frontal and temporal cortices, insula and thalamus. Both VBM and ROI analyses revealed non-overlapping changes in multiple regions, including cerebellum and vermis, bilateral mid frontal, mid occipital cortices, left inferior temporal and precentral gyri. These findings highlight the importance of accounting for sex differences in cross-sectional analyses, not only in the study of normative changes, but particularly in the context of psychiatric and neurologic disorders, wherein sex effects may be confounded with disease-related changes.

The nicotinamide phosphoribosyltransferase antagonist FK866 inhibits growth of prostate tumour spheroids and increases doxorubicin retention without changes in drug transporter and cancer stem cell protein expression.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD) synthesis and is involved in cancer cell proliferation through regulation of energy production pathways. Therefore, NAMPT inhibitors are promising drugs for cancer therapy by limiting energy supply of tumours. Herein, we demonstrated that the NAMPT inhibitor FK866 ((E)-N-(4-(1-Benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) dose-dependently inhibited growth and cell motility of DU-145 prostate tumour spheroids and decreased the intracellular ATP concentration. The apoptosis marker cleaved caspase-3 remained unchanged, but the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) was upregulated. Growth inhibition was reversed upon co-administration of NAD to the cell culture medium. FK866 decreased calcein as well as pheophorbide A efflux from tumour spheroids and increased doxorubicin toxicity, indicating interference with function of drug efflux transporters. DU-145 multicellular tumour spheroids expressed the stem cell associated markers CD133, CD44, Oct4, Nanog, Sox2, and drug transporters ABCB1, ABCG2, and ABCC1 which are associated with stem cell properties in cancer cells. The ABCB1 inhibitor zosuquidar, the ABCG2 inhibitor Ko143, and the ABCC1 inhibitor MK571 increased calcein retention. Neither protein expression of stem cell markers, nor drug transporters was significantly changed upon FK866 treatment. In conclusion, our data suggest that FK866 inhibits prostate cancer cell proliferation by interference with the energy metabolism, and function of drug efflux transporters.

Zoxazolamine-induced stimulation of cardiomyogenesis from embryonic stem cells is mediated by Ca2+, nitric oxide and ATP release.

Ca2+-activated potassium (KCa) channels of small and intermediate conductance influence proliferation, apoptosis, and cell metabolism. We analysed whether prolonged activation of KCa channels by zoxazolamine (ZOX) induces differentiation of mouse embryonic stem (ES) cells towards cardiomyocytes. ZOX treatment of ES cells dose-dependent increased the number and diameter of cardiac foci, the frequency of contractions as well as mRNA expression of the cardiac transcription factor Nkx-2.5, the cardiac markers cardiac troponin I (cTnI), α-myosin heavy chain (α-MHC), ventricular myosin light chain-2 (MLC2v), and the pacemaker hyperpolarization-activated, cyclic nucleotide-gated 4 channel (HCN4). ZOX induced hyperpolarization of membrane potential due to activation of IKCa, raised intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) in a Ca2+-dependent manner. The Ca2+ response to ZOX was inhibited by chelation of Ca2+ with BAPTA-AM, release of Ca2+ from intracellular stores by thapsigargin and the phospholipase C (PLC) antagonist U73,122. Moreover, the ZOX-induced Ca2+ response was blunted by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) as well as the specific P2Y1 antagonist MRS 2,179, suggesting purinergic receptor-stimulated signal transduction. Consequently, ZOX initiated ATP release from differentiating ES cells, which was inhibited by the chloride channel inhibitor NPPB and the gap junction inhibitor carbenoxolone (CBX). The stimulation of cardiomyogenesis by ZOX was blunted by the nitric oxide synthase (NOS) inhibitor l-NAME, as well as CBX and NPPB. In summary, our data suggest that ZOX enhances cardiomyogenesis of ES cells by ATP release presumably through gap junctional hemichannels, purinergic receptor activation and intracellular Ca2+ response, thus promoting NO generation.

Omega-3 and Omega-6 polyunsaturated fatty acids stimulate vascular differentiation of mouse embryonic stem cells.

Polyunsaturated fatty acids (PUFAs) and their metabolites may influence cell fate regulation. Herein, we investigated the effects of linoleic acid (LA) as ω-6 PUFA, eicosapentaenoic acid (EPA) as ω-3 PUFA and palmitic acid (PA) on vasculogenesis of embryonic stem (ES) cells. LA and EPA increased vascular structure formation and protein expression of the endothelial-specific markers fetal liver kinase-1, CD31 as well as VE-cadherin, whereas PA was without effect. LA and EPA increased reactive oxygen species (ROS) and nitric oxide (NO), activated endothelial NO synthase (eNOS) and raised intracellular calcium. The calcium response was inhibited by the intracellular calcium chelator BAPTA, sulfo-N-succinimidyl oleate which is an antagonist of CD36, the scavenger receptor for fatty acid uptake as well as by a CD36 blocking antibody. Prevention of ROS generation by radical scavengers or the NADPH oxidase inhibitor VAS2870 and inhibition of eNOS by L-NAME blunted vasculogenesis. PUFAs stimulated AMP activated protein kinase-α (AMPK-α) as well as peroxisome proliferator-activated receptor-α (PPAR-α). AMPK activation was abolished by calcium chelation as well as inhibition of ROS and NO generation. Moreover, PUFA-induced vasculogenesis was blunted by the PPAR-α inhibitor GW6471. In conclusion, ω-3 and ω-6 PUFAs stimulate vascular differentiation of ES cells via mechanisms involving calcium, ROS and NO, which regulate function of the energy sensors AMPK and PPAR-α and determine the metabolic signature of vascular cell differentiation.

Regulation of somatostatin expression by vitamin D3 and valproic acid in human adipose-derived mesenchymal stem cells.

BACKGROUND: Adipose-derived mesenchymal stem cells (ADMSC) are non-haematopoietic, fibroblast-like multipotent progenitor cells. They have the potential for trilineage (adipocyte, chondrocyte and osteocyte) differentiation as well as differentiation into endocrine pancreatic progenitors. In diabetic or cancer therapy, somatostatin (SST) expression plays a vital role. Small molecules such as valproic acid (VPA) and micronutrients like vitamin D3 have differentiation potential in ADMSC. Therefore, the aim of this study was to investigate the role of vitamin D3 machinery and its metabolic enzymes in ADMSC. Furthermore, the reprogramming effect of vitamin D3 and VPA was evaluated on somatostatin expression in pancreatic lineage differentiation. METHODS: ADMSC were characterised based on their cell surface marker profile using flow cytometry. Specific adipogenic and osteogenic differentiation protocols were used in this study. Gene expression of several pluripotent, endodermal, pancreatic progenitor and pancreatic endocrine lineage markers were investigated in native ADMSC and after stimulation with different concentration of vitamin D3 for five consecutive days (0, 50, 100, 150 nM) and VPA (0.5, 1, 1.5, 2 mM) by real-time PCR. Furthermore, somatostatin expression was confirmed with ELISA and immunocytochemistry. RESULTS: In ADMSC, the expression of somatostatin mRNA, the vitamin D receptor (VDR) and its metabolising enzymes 1 α-Hydroxylase, 24-Hydroxylase and 25-Hydroxylase were detected. Upon stimulation with vitamin D3, nuclear translocation of vitamin D receptor (VDR) was observed. Interestingly, the presence of vitamin D3 reduced the transcription of the somatostatin gene. By contrast, VPA treatment of cultivated ADMSC showed enhancing effect on somatostatin gene expression. No other pluripotent, endodermal, pancreatic progenitor or pancreatic endocrine lineage mRNA expression was modulated under the influence of vitamin D3 and VPA. CONCLUSION: Human ADMSC carry the VDR. The vitamin D metabolising enzyme 25-Hydroxylase responded to the addition of vitamin D3. Moreover, our results demonstrate that somatostatin expression in ADMSC is constitutive, partially secreted and regulated by vitamin D3 and VPA.

Stigma experiences and perceived stigma in patients with first-episode schizophrenia in the course of 1 year after their first in-patient treatment.

Patients with schizophrenia suffer from stigma and discrimination due to their illness. Yet it is not well examined how experiences of stigma and discrimination express at the early illness stage and how they develop subsequently. Therefore, clinical and psycho-social correlates of stigma experiences and perceived stigma are analyzed in patients with first-episode schizophrenia over the course of 1 year after their first in-patient treatment. Questionnaire data assessed within the multi-centre-RCT "First-Episode Study" of the German Research Network on Schizophrenia were analyzed. Patients with first-episode schizophrenia were assessed 8 weeks after their first in-patient treatment (post-acute assessment) and 1 year later. N = 48 (post-acute) and N = 24 (1-year follow-up) patients provided questionnaire data appropriate for analyses, with N = 12 dyads. These data included burden due to stigma experiences (B-STE), perceived stigma (PDDQ), clinical (PANSS, CDSS, CGI, GAF, SAS) and psycho-social factors (LQLP, FSNK-self-esteem, KK-Scale). Cross-lag-correlation models showed a causal relation between stigma experiences (post-acute) and reduced self-esteem after 1 year. Multiple regression models revealed different models for experienced and perceived stigma. Factors associated with higher stigma experiences were older age, worse clinical global impression, better social adjustment, lower self-esteem, and the belief that illness is not driven by chance or fate. The different associations between psycho-social factors and stigma experiences and perceived stigma demonstrate the complexity of this inter-relationship. The results have practical implications for psycho-educational and other therapeutic interventions addressing stigma coping. Since the sample was small and selective, replication studies are needed.

The milk thistle (Silybum marianum) compound Silibinin stimulates leukopoiesis from mouse embryonic stem cells.

The milk thistle compound Silibinin (i.e., a 1:1 mixture of Silybin A and Silybin B) stimulates vasculogenesis of mouse embryonic stem (ES) cells. Because vasculogenesis and leukopoiesis are interrelated, the effect of Silibinin on leukopoiesis of ES cells was investigated. Treatment of differentiating ES cells with hydrosoluble Silibinin-C-2',3-dihydrogen succinate dose-dependent increased the number of CD18+ , CD45+ , and CD68+ cells, indicating leukocyte/macrophage differentiation. Silibinin treatment activated phosphoinositide 3-kinase (PI3K), AKT (protein kinase B), signal transducer and activator of transcription 3 (STAT3), stimulated hypoxia-induced factor-1α (HIF-1α), and vascular endothelial growth factor receptor 2 (VEGFR2) expression and raised intracellular nitric oxide (NO). Western blot experiments showed that upon coincubation with either the PI3K inhibitor LY294002, the STAT3 inhibitor Stattic, the AKT antagonist AKT inhibitor VIII, or the NO inhibitor L-NAME, the Silibinin-induced expression of CD18, CD45, and CD68 was abolished. Moreover, the stimulation of HIF-1α and VEGFR2 expression was blunted upon STAT3 and PI3K/AKT inhibition. Treatment of differentiating ES cells with L-NAME abolished the stimulation of VEGFR2 and VE-cadherin expression achieved with Silibinin, indicating that NO is involved in vasculogenesis and leukocyte differentiation pathways. In summary, the data of the present study demonstrate that Silibinin stimulates leukocyte differentiation of ES cells, which is associated to vasculogenesis and regulated by PI3K/AKT-, STAT3-, and NO-mediated signaling.

Erratum: Mitochondrial G8292A and C8794T mutations in patients with Niemann-Pick disease type C.

[This corrects the article DOI: 10.3892/br.2018.1095.].

Mitochondrial G8292A and C8794T mutations in patients with Niemann-Pick disease type C.

Niemann-Pick disease type C (NP-C) is a neurovisceral lipid storage disorder. At the cellular level, the disorder is characterized by accumulation of unesterified cholesterol and glycolipids in the lysosomal/late endosomal system. NP-C is transmitted in an autosomal recessive manner and is caused by mutations in either the NPC1 (95% of families) or NPC2 gene. The estimated disease incidence is 1 in 120,000 live births, but this likely represents an underestimate, as the disease may be under-diagnosed due to its highly heterogeneous presentation. Variants of adenosine triphosphatase (ATPase) subunit 6 and ATPase subunit 8 (ATPase6/8) in mitochondrial DNA (mtDNA) have been reported in different types of genetic diseases including NP-C. In the present study, the blood samples of 22 Iranian patients with NP-C and 150 healthy subjects as a control group were analyzed. The DNA of the blood samples was extracted by the salting out method and analyzed for ATPase6/8 mutations using polymerase chain reaction sequencing. Sequence variations in mitochondrial genome samples were determined via the Mitomap database. Analysis of sequencing data confirmed the existence of 11 different single nucleotide polymorphisms (SNPs) in patients with NP-C1. One of the most prevalent polymorphisms was the A8860G variant, which was observed in both affected and non-affected groups and determined to have no significant association with NP-C incidence. Amongst the 11 polymorphisms, only one was identified in the ATPase8 gene, while 9 including A8860G were observed in the ATPase6 gene. Furthermore, two SNPs, G8292A and C8792A, located in the non-coding region of mtDNA and the ATPase6 gene, respectively, exhibited significantly higher prevalence rates in NP-C1 patients compared with the control group (P<0.01). The present study suggests that there may be an association between mitochondrial ATPase6/8 mutations and the incidence of NP-C disease. In addition, the mitochondrial SNPs identified maybe pathogenic mutations involved in the development and prevalence of NP-C. Furthermore, these results suggest a higher occurrence of mutations in ATPase6 than in ATPase8 in NP-C patients.

Silibinin from Silybum marianum Stimulates Embryonic Stem Cell Vascular Differentiation via the STAT3/PI3-K/AKT Axis and Nitric Oxide.

Silibinin, the bioactive compound of milk thistle (Silybum marianum), exerts tissue protective and regenerative effects that may include stem cell differentiation toward vascular cells. The purpose of the present study was to investigate whether silibinin stimulates blood vessel formation from mouse embryonic stem (ES) cells and to unravel the underlying signaling cascade. Vascular branching points were assessed by confocal laser scanning microscopy and computer-assisted image analysis of CD31-positive cell structures. Protein expression of vascular markers and activation of protein kinases were determined by western blot. Nitric oxide (NO) generation was investigated by use of the fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. Silibinin dose-dependently increased CD31-positive vascular branching points in embryoid bodies cultivated from ES cells. This was paralleled by increase of protein expression levels for the endothelial-specific markers vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor 2, and hypoxia-inducible factor-1α. Moreover, silibinin increased activation of endothelial nitric oxide synthase (eNOS), which boosted generation of NO in embryoid bodies and enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphoinositide 3-kinase (PI3-K) and AKT. Vasculogenesis, VE-cadherin expression, STAT3 and AKT phosphorylation, NO generation, and eNOS phosphorylation were inhibited by the small molecule STAT3 inhibitor Stattic, AKT inhibitor VIII, the PI3-K inhibitor LY294002, or the NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride. In conclusion, our findings indicate that silibinin induces vasculogenesis of ES cells via activation of STAT3, PI3-K, and AKT, which regulate NO generation by eNOS.

The Milk Thistle (Silybum marianum) Compound Silibinin Inhibits Cardiomyogenesis of Embryonic Stem Cells by Interfering with Angiotensin II Signaling.

The milk thistle (Silybum marianum (L.) Gaertn.) compound silibinin may be an inhibitor of the angiotensin II type 1 (AT1) receptor which is expressed in differentiating embryonic stem (ES) cells and is involved in the regulation of cardiomyogenesis. In the present study, it was demonstrated that silibinin treatment decreased the number of spontaneously contracting cardiac foci and cardiac cell areas differentiated from ES cells as well as contraction frequency and frequency of calcium (Ca2+) spiking. In contrast, angiotensin II (Ang II) treatment stimulated cardiomyogenesis as well as contraction and Ca2+ spiking frequency, which were abolished in the presence of silibinin. Intracellular Ca2+ transients elicited by Ang II in rat smooth muscle cells were not impaired upon silibinin treatment, excluding the possibility that the compound acted on the AT1 receptor. Ang II treatment activated extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways in embryoid bodies which were abolished upon silibinin pretreatment. In summary, our data suggest that silibinin inhibits cardiomyogenesis of ES cells by interfering with Ang II signaling downstream of the AT1 receptor.

State marker properties of niacin skin sensitivity in ultra-high risk groups for psychosis - An optical reflection spectroscopy study.

Impaired niacin sensitivity (NS) is one of the most replicated findings in untreated schizophrenia, and reflects a disturbance of prostaglandin-mediated pathways in association with deregulated arachidonic acid metabolism, pro-inflammatory activation, and vasomotor function. In ultra-high risk individuals (UHR) increased NS was reported recently, pointing towards dynamic alterations of the underlying pathomechanisms in the period preceding psychosis. However, these characteristics are still unresolved in the diverse UHR groups. We tested the hypothesis that NS is attenuated in patients who have transitioned to psychosis and in the Brief Limited Intermittent Psychotic Symptoms (BLIPS, UHR-B) and/or the attenuated symptoms (UHR-A) groups, while it is unchanged or increased in the genetic risk group (UHR-G). Sensitivity to three concentrations (0.1-0.001M) of aqueous methylnicotinate was tested in 84 UHR patients, 105 first-episode psychosis patients (FEP) and 180 healthy individuals (HC), using optical reflection spectroscopy (ORS). The UHR subgroup and transition/non-transition outcomes were assessed according to PACE criteria using the CAARMS. Psychopathology was assessed using SANS, SAPS, and BPRS or SCL-90-R self-ratings. In 0.001M data, decreased NS was found in the UHR-B (n=12), UHR-A (n=45) and the transition groups (n=13), similar to the result in FEP. NS in the UHR-G (n=27) and HC groups did not differ. In the UHR-B and FEP groups, NS and positive symptom scores were inversely correlated. These state marker properties could be used to characterize the intensity of the underlying pathomechanisms during the onset of psychosis or to identify UHR individuals that might benefit from related indicated prevention strategies.

Common and distinct structural features of schizophrenia and bipolar disorder: The European Network on Psychosis, Affective disorders and Cognitive Trajectory (ENPACT) study.

INTRODUCTION: Although schizophrenia (SCZ) and bipolar disorder (BD) share elements of pathology, their neural underpinnings are still under investigation. Here, structural Magnetic Resonance Imaging (MRI) data collected from a large sample of BD and SCZ patients and healthy controls (HC) were analyzed in terms of gray matter volume (GMV) using both voxel based morphometry (VBM) and a region of interest (ROI) approach. METHODS: The analysis was conducted on two datasets, Dataset1 (802 subjects: 243 SCZ, 176 BD, 383 HC) and Dataset2, a homogeneous subset of Dataset1 (301 subjects: 107 HC, 85 BD and 109 SCZ). General Linear Model analyses were performed 1) at the voxel-level in the whole brain (VBM study), 2) at the regional level in the anatomical regions emerged from the VBM study (ROI study). The GMV comparison across groups was integrated with the analysis of GMV correlates of different clinical dimensions. RESULTS: The VBM results of Dataset1 showed 1) in BD compared to HC, GMV deficits in right cingulate, superior temporal and calcarine cortices, 2) in SCZ compared to HC, GMV deficits in widespread cortical and subcortical areas, 3) in SCZ compared to BD, GMV deficits in insula and thalamus (p<0.05, cluster family wise error corrected). The regions showing GMV deficits in the BD group were mostly included in the SCZ ones. The ROI analyses confirmed the VBM results at the regional level in most of the clusters from the SCZ vs. HC comparison (p<0.05, Bonferroni corrected). The VBM and ROI analyses of Dataset2 provided further evidence for the enhanced GMV deficits characterizing SCZ. Based on the clinical-neuroanatomical analyses, we cannot exclude possible confounding effects due to 1) age of onset and medication in BD patients, 2) symptoms severity in SCZ patients. CONCLUSION: Our study reported both shared and specific neuroanatomical characteristics between the two disorders, suggesting more severe and generalized GMV deficits in SCZ, with a specific role for insula and thalamus.